Intranuclear inclusions in the developing neurons of the rat cuneate nuclei

Summary The ultrastructure of intranuclear rodlets, microtubules, fibrillar lattices and membranous inclusions found in the developing cuneate nuclei of rats is described. Rodlets, ranging in diameter from 96–312 nm and in length from 1–2 m, are made up of tightly packed straight filaments measuring 5–8 nm in diameter. Microtubules with a diameter of 26 nm are clustered together. Fibrillar lattices are made up of fibrils with a diameter of 9 nm arranged in layers or sets. Two to nine sets make up a lattice, with a maximum width of 68 nm, in which the adjacent sets are arranged at an angle to each other. Rodlets and fibrillar lattices occur in 6.8% of the neurons. Membranous inclusions, reported here for the first time in normal neurons, are of 2 types: small vesicles of 0.1–0.6 m and large vacuoles measuring 1–2 m. Both types are bounded by either a single or a double membrane and generally have an electron lucent content. Membranous inclusions occur in 25.3 % of the neurons. Changes in the frequency of occurrence of the various intranuclear inclusions in the course of postnatal development are also reported.     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

Efficient Organic Solar Cell with 16.88% Efficiency Enabled by Refined Acceptor Crystallization and Morphology with Improved Charge Transfer and Transport Properties

Single‐layered organic solar cells (OSCs) using nonfullerene acceptors have reached 16% efficiency. Such a breakthrough has inspired new sparks for the development of the next generation of OSC materials. In addition to the optimization of electronic structure, it is important to investigate the essential solid‐state structure that guides the high efficiency of bulk heterojunction blends, which provides insight in understanding how to pair an efficient donor–acceptor mixture and refine film morphology. In this study, a thorough analysis is executed to reveal morphology details, and the results demonstrate that Y6 can form a unique 2D packing with a polymer‐like conjugated backbone oriented normal to the substrate, controlled by the processing solvent and thermal annealing conditions. Such morphology provides improved carrier transport and ultrafast hole and electron transfer, leading to improved device performance, and the best optimized device shows a power conversion efficiency of 16.88% (16.4% certified). This work reveals the importance of film morphology and the mechanism by which it affects device performance. A full set of analytical methods and processing conditions are executed to achieve high efficiency solar cells from materials design to device optimization, which will be useful in future OSC technology development.     

A Site‐Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9‐Mediated Genome Editing Strategies in CHO Cells

Targeted gene knockout and site‐specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells are characterized by low efficiencies. Moreover, numerous strategies proposed to boost SSI efficiency in mammalian cell types have yet to be evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, the SSIGNAL (s ite‐s pecific i ntegration and g en ome al teration) reporter system is developed. This tool can analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR‐associated protein 9)‐mediated disruption activity alone or in conjunction with SSI efficiency. The reporter system uses green and red dual‐fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, two of its applications are demonstrated by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology.     

The use and misuse of regression models in landscape genetic analyses

The field of landscape genetics has been rapidly evolving, adopting and adapting analytical frameworks to address research questions. Current studies are increasingly using regression‐based frameworks to infer the individual contributions of landscape and habitat variables on genetic differentiation. This paper outlines appropriate and inappropriate uses of multiple regression for these purposes, and demonstrates through simulation the limitations of different analytical frameworks for making correct inference. Of particular concern are recent studies seeking to explain genetic differences by fitting regression models with effective distance variables calculated independently on separate landscape resistance surfaces. When moving across the landscape, organisms cannot respond independently and uniquely to habitat and landscape features. Analyses seeking to understand how landscape features affect gene flow should model a single conductance or resistance surface as a parameterized function of relevant spatial covariates, and estimate the values of these parameters by linking a single set of resistance distances to observed genetic dissimilarity via a loss function. While this loss function may involve a regression‐like step, the associated nuisance parameters are not interpretable in terms of organismal movement and should not be conflated with what is actually of interest: the mapping between spatial covariates and conductance/resistance. The growth and evolution of landscape genetics as a field has been rapid and exciting. It is the goal of this paper to highlight past missteps and demonstrate limitations of current approaches to ensure that future use of regression models will appropriately consider the process being modeled, which will provide clarity to model interpretation.     

“Reverse” Carbocyclic Fleximers: Synthesis of a New Class of Adenosine Deaminase Inhibitors

A series of flexible carbocyclic pyrimidine nucleosides has been designed and synthesized. In contrast to previously reported “fleximers” from our laboratory, these analogues have the connectivity of the heterocyclic base system “reversed”, where the pyrimidine ring is attached to the sugar moiety, rather than the five membered imidazole ring. As was previously seen with the ribose fleximers, their inherent flexibility should allow them to adjust to enzyme binding site mutations, as well as increase the affinity for atypical enzymes. Preliminary biological screening has revealed surprising inhibition of adenosine deaminase, despite their lack of resemblance to adenosine.     

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates